GENETIC REGUIATORY EVENTS IN T LYMPHOCYTE ACTIVATION 
Gerald R. Crabtree, M.D., Associate Investigator 
Work in the Crabtree laboratory centers around 
molecular mechanisms of cellular differentiation. 
The fundamental events involved in this process 
are studied using two systems: T lymphocyte activa- 
tion and the developmental control of the hepatic 
phenotype. Work on T lymphocyte activation is 
funded by the Hov^^ard Hughes Medical Institute. 
T lymphocyte activation refers to the sequential 
morphologic and functional changes that culminate 
in cell proliferation and immune function after a 
quiescent T cell is exposed to its cognate antigen in 
the proper histocompatibility context. Because 
early studies indicated that only 30-60 min of ex- 
posure to lectins is required for commitment to T 
cell activation, attention has been focused on these 
early events after antigen interacts with the antigen 
receptor. The hypothesis that guides this work is 
that essential genetic programming events are set in 
motion during this time. Understanding the molec- 
ular nature of these programming events is the ob- 
ject of these studies. 
I. Identification of Nuclear Targets for the T Lym- 
phocyte Antigen Receptor. 
Because signals from the antigen receptor govern 
the decision to initiate the activation pathway or re- 
main in a quiescent state, Dr. Crabtree and his col- 
leagues began a number of years ago to define nu- 
clear targets for the antigen receptor. They chose 
the interleukin-2 (IL-2) gene to search for such nu- 
clear targets, since 1) the IL-2 gene is rigorously 
controlled by the antigen receptor, 2) expression of 
IL-2 is essential for proliferation and immunologic 
activation, and 3) transcription of the gene is initi- 
ated at the time that T cells become committed to 
proliferation and immunologic function. These 
studies led to the identification of a transcriptional 
enhancer lying just 5' of the IL-2 gene that medi- 
ated T cell specificity, antigen-receptor inducibility, 
and inhibition by cyclosporin and glucocorticoids. 
The precise sequences required for the action of 
this enhancer were defined by examining the func- 
tion of a series of linker-scanner and internal-dele- 
tion mutants within the enhancer transfected into 
T cell lines. These studies led to the recognition of 
three short sequences of 10-20 bp that were essen- 
tial for full function of the enhancer. The role of 
these sequences in the function of the IL-2 en- 
hancer was supported by studies in which these se- 
quences were synthesized and several copies at- 
tached to an unrelated promoter. Two of these syn- 
thetic sequences, in two, three, or four copies, acti- 
vate transcription of an unrelated promoter in 
response to signals from the T cell antigen recep- 
tor; hence they were called antigen receptor re- 
sponse elements (ARRE-1 and ARRE-2). 
A. A novel function for the Oct-1 transcription fac- 
tor in T cell activation. The most 3 -proximal 
ARRE binds a protein found in all cell types exam- 
ined to date. The protein binding to this site was 
initially called NFIL2-A. More recently, this protein 
has been purified and found to have the same mo- 
lecular weight, sequence specificity, and antigenic 
determinants as the previously identified Oct-1 
transcription factor. In addition, proteolytic diges- 
tion fragments generated with V-8 protease and Arg 
C protease are identical, strongly indicating that 
NFIL2-A is identical to Oct-1. Because tandem re- 
peats of this site activate transcription within 15-20 
min after triggering the antigen receptor and activa- 
tion by this site does not require protein synthesis, 
the group now believes that the Oct-1 transcription 
factor is post-translationally modified or binds a 
second protein that is post-translationally modified. 
These results, which indicate that Oct-1 is transmit- 
ting signals directly from the antigen receptor, will 
facilitate the approach to understanding how sig- 
nals from the antigen receptor are transmitted to 
the nucleus. In the coming year the major objective 
of the laboratory will be to characterize the way in 
which the Oct-1 protein becomes active in re- 
sponse to signals initiated at the antigen receptor. 
B. NFAT protein appears to account for the tissue 
specificity and protein synthetic requirement for 
early gene activation. The most 5' functional site 
in the IL-2 enhancer binds a protein that has been 
found only in nuclear extracts of activated T cells. 
This protein, nuclear factor of activated T cells 
(NFAT), appears —15-20 min after T cells are acti- 
vated with lectin and precedes the first detection of 
IL-2 mRNA by 10-20 min. The appearance of the 
binding activity requires the activation of an earlier 
gene. In footprinting and competition studies, 
NFAT or a very similar protein binds to sequences 
important for transcriptional activation of the long- 
terminal repeat (LTR) of human immunodeficiency 
virus (HIV). Thus NFAT-1 may be a general regula- 
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